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RESEARCH ARTICLE Effect of Withaferin a on A549 Cellular DOI:http://dx.doi.org/10.7314/APJCP.2014.15.4.1711 Effects of Withaferin A on A549 Cellular Proliferation and Apoptosis in Non-small Cell Lung Cancer Cells RESEARCH ARTICLE Effect of Withaferin A on A549 Cellular Proliferation and Apoptosis in Non-small Cell Lung Cancer Yong Cai1, Zhao-Ying Sheng1, Yun Chen1, Chong Bai2* Abstract Objective: To explore the effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer (NSCLC). Materials and Methods: NSCLC cell line A549 was selected to explore the effect of Withaferin A on A549 cellular proliferation, apoptosis and the PI3K/Akt signal pathway capable of regulating tumor biological behavior by assessment of cellular proliferation, cellular apoptotic rates and cellular cycling as well as by immuno-blotting. Results: Withaferin A could inhibit A549 cellular proliferation and the control rate was dosage-dependent (P<0.05), which also increased time-dependently with the same dosage of Withaferin A (P<0.05). The apoptotic indexes in A549 cells treated with 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A for 48 h were significantly different (P<0.05). In addition, the apoptotic rates of each group in both early and advanced stages were higher than those in 0 μmol·L-1 (P<0.05), which were evidently higher after 48 h than those after 24 h (P<0.05). A549 cells treated by Withaferin A for 48 h were markedly lower in Bcl-2 level and obviously higher in Bax and cleaved caspase-3 levels than those treated by 0 μmol·L-1 Withaferin A (P<0.05), and there were significant differences among 5, 10 and 20 μmol·L-1 Withaferin A (P<0.05). The ratios of A549 cells -1 treated by Withaferin A for 48 h in G0/G1 stage were higher than those in 0 μmol·L , while those in S and G2/M stages were obviously lower than those in G2/M stage, and there were significant differences in 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A (P<0.05). Additionally, p-Akt/Akt values were in reverse association with dosage, and the differences were significant P( <0.05). Conclusion: Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing activation of the PI3K/Akt pathways. Keywords: Non-small cell lung cancer - proliferation - apoptosis - A549 cells Asian Pac J Cancer Prev, 15 (4), 1711-1714 Introduction proliferation, apoptosis and PI3K/Akt signal pathways capable of regulating tumor biological behaviors, and the Lung cancer is the leading cause for tumor-associated results are as follows. deaths, in which non-small cell lung cancer (NSCLC) accounts for 85% and most patients are in advanced stages Materials and Methods with poor therapeutic efficacy when diagnosed (Jemal et al., 2010; Cho et al.,2013; Lu et al., 2013; Wang et Agents al., 2013; Yan et al., 2013; Kim et al., 2014). Though A549 cell lines were obtained from American ATCC; there has been great development in clinical treatment of Withaferin A, dimethyl sulfoxide (DMSO), methyl NSCLC, its prognosis is still unsatisfactory due to the low thiazolyl tetrazolium (MTT), Sodium dodecyl sulfate rate of 5-year survival in clinic (Verdecchia et al., 2007). (SDS) and Tris were purchased from American Sigma- Therefore, seeking more effective drugs for NSCLC is of Aldrich company; RPMI 1640 culture medium, Fetal calf great significance in its clinical treatment. Withaferin A is Serum (FCS) and trypsase were brought from American an active ingredient extracted from Withania omnifera (Yu Gibco company; penicillin and Streptomycin were gained et al., 2013), which possesses the functions of regulating from Hyclone company; glycine, RNase, Acrylamide and immunity and anti-inflammation, etc.. In recent years, it methylene-bisacrylamide were received from American has also been discovered that Withaferin A has anti-tumor Amresco company; Annexin-FITC/PI kits for apoptotic activity and is effective in inhibiting multiple tumors, detection was brought from Keygen Biotech. Co., Ltd; such as breast cancer, etc. (Hahm et al., 2013; Lee et al., internal references GAPDH, Bcl-2 and Cleaved caspase-3 2013). NSCLC cell line A549 was selected in this study polyclonal antibodies were obtained from American to explore the effect of Withaferin A on A549 cellular Cell Sigaling Technology company; Bax, Akt and P-Akt 1Department of Radiation Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 2Department of Respiration, Changhai Hospital, Second Military Medical University, Shanghai, China *For correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 1711 Yong Cai et al Table 1. Effect of Withaferin A on Cellular Apoptosis (%, χ±s) Groups 24 h 48 h Early stage Advanced stage Early stage Advanced stage 0 μmol·L-1 3.18±0.53 1.16±0.73 4.42±1.26 2.21±0.93 2.5 μmol·L-1 6.72±1.90a 3.25±1.19a 11.63±2.48ab 6.38±1.42ab 5.0 μmol·L-1 11.91±2.91a 6.84±1.35a 13.57±2.35a 9.27±1.55ab 10.0 μmol·L-1 17.08±4.18a 8.29±2.51a 22.29±4.70ab 10.65±2.82a 20.0 μmol·L-1 21.37±3.37a 11.68±2.94a 28.68±5.58ab 15.63±2.73ab Compared with 0 μmol·L-1, aP<0.05; Compared with 24 h, bP<0.05 Figure 1. Control Rate of Cellular Proliferation A for 48 h were collected and centrifuged at high-speed, Treated with Different Dosages of Withaferin A supernatant was discarded and cells were decomposed polyclonal antibodies were purchased from Santa Cruz on ice to determine the protein levels by BAC method. company; and Hoechst 33258 was brought from Beyotime The protein samples were mixed with loading buffer and Institute of Biotechnology. given 10% SDS-PAGE gel electrophoresis under routine methods. The constant voltages of spacer and separation Cellular proliferation detection gels were 80 V (for 30 min) and 100 V (about 80 min), A549 cells in logarithmic phase were treated by 0.25% respectively. Then, the samples were transformed to trypsase, made into mono-cellular suspension by blowing PVDF membrane in semi-dry condition, added with and beating, inoculated in 96-well plate by 1×105/well appropriate amounts of p-Akt primary antibodies (1: for totally 200 μL and cultured for 24 h, added with 20 200), Bax, Caspase-3 and Bcl-2 (1: 300), incubated at μL 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A and 4℃ overnight, added with secondary antibodies (1: 3000) cultured for 24, 48, 72 and 96 h respectively in incubators and incubated for 2 h in room temperature by shaking, containing 5% CO2 saturated humidity at 37℃, added with which were colored by ECL and developed to analyze 10 μL 5 mg·mL-1 MTT solution in each well, and cultured the optical intensity of each developed stripe by Gel-Pro for 4 h. Optical density A492 in each well under 492 nm analyzer. p-Akt was the ratio of solved solution with Akt was measured by ELIASA and it was set up as 100% in while the relevant expressions of other proteins were the control group. Three parallel holes were established in ratios with GAPDH. each group and the study was repeated for 3 times to obtain the mean values. Control rate (%)=[ (control group-blank Statistical data analysis group)- (medicine group-blank group)]/ ( control group- Windows SPSS 16.0 software was applied to analyzed blank group)×100%. data expressed by Mean±Standard Deviation (χ±s), while one way ANOVA was used for multi-group comparisons Apoptotic index detection and SNK method for paried-comparison. P<0.05 was A549 mono-cellular suspension was inoculated on regarded as statistically significant. sterilized slide placed in the bottom of 12-well culture plate, and culture medium was discarded after cellular Results adherence presented. Then culture mediums containing different dosages of Withaferin A were added and Effect of Withaferin A on cellular proliferation cultured for 48 h, added with paraformaldehyde to fix A549 cells were treated by 0, 2.5, 5.0, 10.0 and 20.0 cells, washed by PBS, added with 5 μg·mL-1 Hoechst μmol·L-1 Withaferin A for 24 h, 48 h, 72 h and 96 h, 33258 and stained for 30 min at room temperature respectively, and the MTT laboratory results showed that shielded from light. Fluorescent microscope was used Withaferin A could inhibit the proliferation of A549 cells, to observe cellular morphology and 5 high-power fields which was in positive association with the dosages and in were selected to calculate the counts of apoptotic cells and dosage-dependent relationship (P<0.05). In addition, in total cells, so as to obtain the apoptotic indexes. Apoptotic the same dosage of Withaferin A, the cellular control rate index=Apoptotic cell count/Total cell count×100%. also increased time-dependently (P<0.05) (Figure 1). Detections of cellular apoptotic rate and cellular cycle Effect of Withaferin A on cellular apoptosis A549 cells were inoculated in 6-well culture plate Cellular apoptotic indexes: The apoptotic indexes of by 1×106/well, added with 20 μL 0, 2.5, 5.0, 10.0 and A549 cells treated respectively by 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A and cultured for 24 h in 5% 20.0 μmol·L-1 Withaferin A for 48 h were (2.75±0.64)%, CO2 incubator at 37℃ respectively, washed by PBS (4.61±1.36)%, (9.75±2.78)%, (12.92±3.42)% and once based on the instruments of apoptosis detection kit, (18.68±4.31)% respectively, and the differences were all added with Annexin V/FITC and PI, and stained for 15 significant (P<0.05) (Figure 2).
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